Jundishapur J Microbiol. 2015 October; 8(10): e22249.
doi: 10.5812/jjm.22249
Research Article
Published online 2015 October 12.
Comparative Analysis of Denaturing Gradient Gel Electrophoresis and
Temporal Temperature Gradient Gel Electrophoresis Profiles as a Tool for
the Differentiation of Candida Species
1,*
2
2
Parisa Mohammadi, Aida Hamidkhani, and Ezat Asgarani
1Department of Microbiology, Faculty of Biological Sciences, Alzahra University, Tehran, IR Iran
2Department of Biotechnology, Faculty of Biological Sciences, Alzahra University, Tehran, IR Iran
*Corresponding author: Parisa Mohammadi, Department of Microbiology, Faculty of Biological Sciences, Alzahra University, Tehran, IR Iran. Tel: +98-2185692714, Fax: +98-2188058912,
E-mail: [email protected]
Received 2014 July 27; Revised 2015 January 19; Accepted 2015 March 1.
Background: Candida species are usually opportunistic organisms that cause acute to chronic infections when conditions in the host
are favorable. Accurate identification of Candida species is an essential pre-requisite for improved therapeutic strategy. Identification of
Candida species by conventional methods is time-consuming with low sensitivity, yet molecular approaches have provided an alternative
way for early diagnosis of invasive candidiasis. Denaturing gradient gel electrophoresis (DGGE) and temporal temperature gradient gel
electrophoresis (TTGE) are polymerase chain reaction (PCR)-based approaches that are used for studying the community structure of
microorganisms. By using these methods, simultaneous identification of multiple yeast species will be possible and reliable results will
be obtained quickly.
Objectives: In this study, DGGE and TTGE methods were set up and evaluated for the detection of different Candida species, and their
results were compared.
Materials and Methods: Five different Candida species were cultured on potato dextrose agar medium for 24 hours. Next, total DNA was
extracted by the phenol-chloroform method. Two sets of primers, ITS3-GC/ITS4 and NL1-GC/LS2 were applied to amplify the desired regions.
The amplified fragments were then used to analyze DGGE and TTGE profiles.
Results: The results showed that NL1-GC/LS2 primer set could yield species-specific amplicons, which were well distinguished and allowed
better species discrimination than that generated by the ITS3-GC/ITS4 primer set, in both DGGE and TTGE profiles. All five Candida species
were discriminated by DGGE and TTGE using the NL1-GC/LS2 primer set.
Conclusions: Comparison of DGGE and TTGE profiles obtained from NL1-GC/LS2 amplicons exhibited the same patterns. Although both
DGGE and TTGE techniques are capable of detecting Candida species, TTGE is recommended because of easier performance and lower
costs.
Keywords: Molecular Assay, Polymerase Chain Reaction-Denaturing Gradient Gel Electrophoresis, Polymerase Chain ReactionTemporal Temperature Gradient Gel Electrophoresis, Candida
1. Background
Candida species are the most common cause of fungal
infections, leading to a range of life threatening and
non-life-threatening diseases. Because of widespread
use of broad-spectrum antibiotics and the growing
numbers of HIV-infected and immune-compromised
patients, the incidence of Candida infections has grown
in the recent years (1). Vaginal candidiasis causes 20 - 25%
of infectious vaginitis cases (2). It is estimated that 75%
of all females experience an episode of vaginal candidiasis in their lifetime (3). Candida albicans, C. glabrata,
C. parapsilosis, and C. tropicalis account for 80 - 90% of
fungal isolates that cause infections worldwide (4). Accurate identification of Candida species is important for
the treatment of infected patients, because significant
attributes such as drug resistance and virulence differ
among species (5).
Commonly, Candida in vaginal samples is identified
by microscopic examination of a wet mount with potassium hydroxide (KOH, amine test). Using this technique
budding yeast cells in only 50 - 70% of females with Candida vaginitis are detected and non-albicans species may
not be identified (6). Alternatively, C. albicans and C. tropicalis can be distinguished by growth on chromogenic
agar medium and other Candida species can be recognized by enzymatic tests. For each of these tests, isolated
organisms have to be grown on solid medium for 24 to 48
hours before the test. In addition, the ‘gold standard’ for
definitive biochemical identification requires analysis of
Copyright © 2015, Ahvaz Jundishapur University of Medical Sciences. This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial 4.0 International License (http://creativecommons.org/licenses/by-nc/4.0/) which permits copy and redistribute the material just in noncommercial usages, provided the original work is properly cited.
Mohammadi P et al.
assimilation and fermentation, taking up to 30 days to
complete (7).
In the recent years, numerous DNA-based techniques
have been developed to improve the identification of
Candida species and provide more accurate results. These
molecular techniques include the polymerase chain reaction (PCR), real-time PCR, restriction fragment length
polymorphism, electrophoretic karyotyping, fluorescence in situ hybridization, randomly amplified polymorphic DNA analysis, multi-locus sequence typing and
pyrosequencing. Each technique has its own advantages
and limitations, and many of them require a culturing
step to isolate the target species (8). Therefore, culture
bias and the loss of minor species can occur. Real-time
PCR method has been developed to detect medically important Candida species in clinical samples (9). This novel
method is sensitive, specific and rapid for Candida detection and estimation, yet it also has its own drawbacks.
The main limitation is its specificity for particular species
or groups of species in a situation where multiple or unpredicted species may occur.
The use of PCR together with denaturing gradient gel
electrophoresis (DGGE) or the related method temporal
temperature gradient gel electrophoresis (TTGE) enables
detection of the presence of such species and frequently
their presumptive identification, even if they are present
as minor populations. In both TTGE and DGGE, DNA fragments of the same length yet with different sequences are
separated, based on decreased electrophoretic mobility
of partially melted double-strand DNA molecules. Separation is performed with polyacrylamide gels containing a
linear gradient of chemical denaturant gradient in DGGE
or a linear temperature gradient in TTGE (10).
2. Objectives
In this study, the ability of DGGE and TTGE techniques
to distinguish different Candida species was assayed and
the results of profiles were compared.
3. Materials and Methods
3.1. Samples and DNA Extraction
Five different standard Candida species including C. albicans (CCUG 32723), C. glabrata (CCUG 35267), C. tropicalis
(CCUG 34274), C. orthopsilosis (CCUG 20503), C. parapsilosis (ATTC 22019), and some clinical strains isolated from
vaginal tracts were analyzed in this study. These strains
were cultured on Potato Dextrose Agar, PDA (Merck, Germany) for 24 hours at 36°C and their genomic DNA was
extracted using the phenol-chloroform method, according to isolation of nucleic acid protocols of yeast (11).
3.2. Polymerase Chain Reaction Conditions
All PCR reactions were performed in a PeqSTAR thermocycler (PEQLAB, Germany). Two primer sets were
2
used. The first primer set was ITS3 and ITS4 (12), and 40
bp GC-clamp was attached to the 5’ end of the forward
primer (13). This primer set amplifies the ITS2 region,
producing amplicons with ~ 300 - 400 bp length. The
second primer set was NL1/LS2 and the 30 bp GC clamp
was attached to the 5’ end of NL1. This primer set amplifies the D1 region of the 26 - 28S rRNA gene, yielding
amplicons of ~ 250 bp length (14). For the first PCR reactions, 5 μL of PCR buffer (CinnaGen, Iran), 1.5 mM of
MgCl2 (CinnaGen, Iran), 0.2 mM of dNTPs (CinnaGen,
Iran), 0.16 mM of each primer (Faza Biotech, Iran), 1.25
U of DNA Taq polymerase (CinnaGen, Iran) and about
20 ng of DNA were used in a final volume of 50 µL. The
amplification program of DNA was started with denaturation for five minutes at 95°C, followed by 35 cycles
of denaturation for 30 seconds at 95°C, primer annealing for 45 seconds at 58°C and extension for one minute
at 72°C. Final extension was at 72°C for five minutes. For
the second PCR, the 50 µL reaction mixture consisted of
5 µL of PCR buffer (CinnaGen, Iran) supplemented with
4 mM MgCl2 (CinnaGen, Iran), 0.2 mM dNTPs (CinnaGen,
Iran), 0.1 mM of each primer (Faza Biotech, Iran), 2.5 U
Taq DNA polymerase (CinnaGen, Iran) and about 20 ng
of DNA. The reactions were performed for 30 cycles. Following an initial four-minute denaturation at 95°C, the
PCR cycle consisted of 95°C for 30 seconds, 53°C for 45
seconds and 72°C for 60 seconds, with a final extension
at 72°C for seven minutes. Polymerase Chain Reaction
products (5 µL) were analyzed on 1% (w/v) agarose gel
(Merck, Germany) by electrophoresis (Bio-Rad, USA).
3.3. Denaturing Gradient Gel Electrophoresis and
Temporal Temperature Gradient Gel Electrophoresis Conditions
The DCode universal mutation detection system (BioRad, Hercules, CA, USA) was used for DGGE and TTGE
analysis. Denaturing Gradient Gel Electrophoresis was
performed using a 1.0 mm polyacrylamide gel [ratio of
8% (w/v) acrylamide (Merck, Germany) to bis-acrylamide
(Merck, Germany), 37.5: 1]. For the ITS3-GC/ITS4 primer PCR
products, 30 - 60% denaturing gradient [100% denaturant
was 7 M urea (Merck, Germany), and 40% (v/v) formamide
(Merck, Germany)], which increased in the direction of
electrophoresis, was applied. The gel was prepared and
run with 1X Tris-acetate-EDTA (TAE) buffer at a constant
voltage of 55 V at 56°C for 16 hours. For primer set NL1-GC/
LS2 amplicons, DGGE was performed with a 30 - 45% denaturing gradient and run with 1X TAE at a constant voltage
of 130 V for 4.5 hours at 60°C.
In TTGE, the polyacrylamide gel composed of 8% (v/v)
acrylamide-bis-acrylamide mixture. Temporal temperature gradient gel electrophoresis was performed at a constant voltage of 65 V and 6 M urea (Merck, Germany) for
14 hours and 17 minutes with a temperature gradient of
56 to 66°C for the ITS2 region and at a constant voltage of
65 V and 7 M urea for 10 hours and 42 minutes with a temJundishapur J Microbiol. 2015;8(10):e22249
Mohammadi P et al.
perature gradient of 51/5 to 62/2°C for the D1 region. The
PCR products (20 μL) were mixed with 20 μL 2x gel loading dye. After electrophoresis, the gels were stained with
ethidium bromide (Merck, Germany) for 30 minutes at
room temperature and visualized by a UV transillumination (Syngen inGenius LHR, UK).
Figure 2. Temporal Temperature Gradient Gel Electrophoresis Profiles
Obtained From Candida Species With the NL1-Gc/Ls2 Primer Set
4. Results
Using the ITS3-GC/ITS4 primer pair for the ITS2 region,
unspecific PCR products were produced, which were not
eliminated by changing PCR conditions, such as number
of cycles, annealing temperature, primer concentration or
amount of template. By using these PCR products in DGGE
and TTGE, appropriate results were not found. Multiple
bands were visualized in the fingerprints derived from a
single Candida species and some of species were not discriminated from each other (data not shown). The NL1-GC/
LS2 primer set produced specific PCR products and by applying these products in DGGE and TTGE for each Candida
species, reproducible clear single bands at a specific position of the gel were achieved (Figures 1 and 2). By comparing clinical samples with standard species, isolated yeasts
were identified as C. albicans and C. glabrata.
Figure 1. Denaturing Gradient Gel Electrophoresis Profiles Obtained
From Candida Species With the NL1-Gc/Ls2 Primer Set
Lane1: C. orthopsilosis (CCUG 20503), lane 2: C. parapsilosis (ATTC 22019),
lane 3: C. tropicalis (CCUG 34274), lane 4: C. glabrata (CCUG 35267), lane 5: C.
albicans (CCUG 32723), lane 6 – 11: clinical samples and lane 12 all standard
species.
5. Discussion
Lane1: C. orthopsilosis (CCUG 20503), lane 2: C. parapsilosis (ATTC 22019),
lane 3: C. tropicalis (CCUG 34274), lane 4: C. glabrata (CCUG 35267), lane 5:
C. albicans (CCUG 32723), lane 6 - 11: clinical samples and lane 12 mixture of
all standard species.
Jundishapur J Microbiol. 2015;8(10):e22249
The incidence of opportunistic Candida infections has
considerably increased in the recent years. Despite progress in the development of new molecular approaches
for the diagnosis of Candida infections, developing a
simple, rapid, and economic effective test for diagnostic
purposes remains elusive. In the present study, DGGE and
TTGE techniques were evaluated to detect different Candida species by using two primer sets, ITS3-GC/ITS4 and
NL1-GC/LS2. These methods are rapid, inexpensive and reliable and allow simultaneous analysis of multiple samples on the same gel (15). Furthermore, DGGE and TTGE
can be used in clinical laboratories to identify pathogenic
Candida species. Weerasekera et al. used the DGGE method for the identification of various Candida species (16)
yet the TTGE technique has not been used for detection of
Candida species so far.
Our results showed that the ITS3-GC/ITS4 primer pair
was not able to produce appropriate PCR products and
yielded multiple banding patterns for some of the species being analyzed by DGGE and TTGE. These findings
are in accordance with other studies, which were carried
out using ITS3-GC/ITS4 primers (17). As the internal tran3
Mohammadi P et al.
scribed spacer (ITS) region is highly variable, production
of multiple bands in DGGE and TTGE may be due to the
sequence variation between different ITS2 copies in the
rRNA operon. Therefore, using the ITS3-GC/ ITS4 primer
pair in DGGE and TTGE may lead to overestimation of microorganism populations being analyzed (18).
Separation of DNA fragments in DGGE and TTGE is based
on decreased electrophoretic mobility of partially melted double-stranded DNA on polyacrylamide gels containing a linear gradient of DNA denaturant in DGGE or
a linear temperature gradient in TTGE. Moreover, DGGE
is complicated by the difficulties of choosing the exact
running time and gel contents to achieve optimal separation. By running a DGGE gel for too long, an achieved
separation decreases, and may even be lost. Furthermore,
gel-casting conditions are not exactly reproducible in
DGGE and it is not possible to compare two different gels.
In contrast, in TTGE, the denaturant concentration and
running time can be determined from theoretical melting curves. Since there is no denaturant gradient, TTGE
gel has higher reproducibility in casting, and easier preparation (19). As it has been shown in the current study by
increasing voltage in DGGE, the electrophoresis time will
be reduced. On the other hand, in TTGE, the electrophoresis time depends on ramp rate (temperature increase
°C/hour). By increasing ramp rate to more than 1°C/hour,
band resolution decreases. Therefore, in the extensive
temperature ranges, the required time for TTGE is more
than DDGE.
In the current study comparison of DGGE and TTGE profiles showed that both of these techniques are capable
to differentiate various Candida species, however, in the
study that was carried out by Farnleitner et al. results
showed that TTGE is not able to separate DNA amplicons
from each other while DGGE was able to do so (20). Our
study exhibited that bands migration for different Candida species has the same patterns in both DGGE and TTGE
profiles, yet resolution of bands in DGGE was better in
comparison with TTGE. As both DGGE and TTGE methods
are based on the same principles, it is expected for their
results to follow the same patterns. However, Marie et al.
reported that DGGE and TTGE patterns appear not to be
directly comparable in the study of picoplankton communities of the Mediterranean Sea (21).
Our data suggested that by using the NL1-GC-LS2 primer
pair, both of DGGE and TTGE techniques are capable to
detect different Candida species, and although TTGE takes
a long time, this method is more appropriate than DGGE
for Candida species due to lower cost, simplicity and reproducibility.
Acknowledgements
The authors thank Maryam Yousefi for her assistance
with the technical methods. All of the microbiological
experiments were carried out at the National Industrial
Microbiology Laboratory of Alzahra University and were
financed by the Vice Chancellor of Alzahra University.
4
Authors’ Contributions
Parisa Mohammadi was responsible for the study concept and design. Acquisition of data was done by Aida
Hamidkhani. Analysis and interpretation of data was
implemented by Aida Hamidkhani, Ezat Asgarani and
Parisa Mohammadi. Aida Hamidkhani prepared the draft
of the manuscript. Critical revision of the manuscript for
important intellectual content as well as supervision of
the study was performed by Parisa Mohammadi. Administrative, technical and material support was provided by
Parisa Mohammadi and Ezat Asgarani.
Funding/Support
This study was supported by the Vice Chancellor of Alzahra University, Tehran, Iran.
References
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
Jarvis WR. Epidemiology of nosocomial fungal infections, with
emphasis on Candida species. Clin Infect Dis. 1995;20(6):1526–30.
Trama JP, Mordechai E, Adelson ME. Detection and identification
of Candida species associated with Candida vaginitis by realtime PCR and pyrosequencing. Mol Cell Probes. 2005;19(2):145–52.
Sobel JD. Pathogenesis and epidemiology of vulvovaginal candidiasis. Ann N Y Acad Sci. 1988;544:547–57.
Pfaller MA. Nosocomial candidiasis: emerging species, reservoirs, and modes of transmission. Clin Infect Dis. 1996;22 Suppl
2:S89–94.
Lopez-Martinez R. Candidosis, a new challenge. Clin Dermatol.
2010;28(2):178–84.
Elliott KA. Managing patients with vulvovaginal candidiasis.
Nurse Pract. 1998;23(3):44–6.
Warren NG, Hazen KC. Candida, Cryptococcus, and other yeasts
of medical importance. In: Murray PR, Barton EJ, Pfaller MA,
Tenover FC, Yolken RH, editors. Manual of clinical microbiology.
Washington, DC: American Society for Microbiology; 1995. p.
723–37.
Trtkova J, Raclavsky V. Molecular-genetic approaches to identification and typing of pathogenic Candida yeasts. Biomed Pap Med
Fac Univ Palacky Olomouc Czech Repub. 2006;150(1):51–61.
Fricke S, Fricke C, Schimmelpfennig C, Oelkrug C, Schonfelder U,
Blatz R, et al. A real-time PCR assay for the differentiation of Candida species. J Appl Microbiol. 2010;109(4):1150–8.
Muyzer G, Smalla K. Application of denaturing gradient gel
electrophoresis (DGGE) and temperature gradient gel electrophoresis (TGGE) in microbial ecology. Antonie Van Leeuwenhoek.
1998;73(1):127–41.
Xiano W. Yeast Protocols. In: Hanna M, Xiano W, editors. Isolation
of Nucleic Acids. Berlin: springer; 2006. pp. 2–4.
White TJ, Bruns T, Lee S, Taylor J. Amplification and Direct Sequencing of Fungal Ribosomal Rna Genes for Phylogenetics. In:
Innis MA, Gelfand DH, Sninsky JJ, White TJ, editors. PCR protocols:
a guide to methods and applications. New York: Academic Press;
1990. p. 315–22.
Muyzer G, de Waal EC, Uitterlinden AG. Profiling of complex microbial populations by denaturing gradient gel electrophoresis
analysis of polymerase chain reaction-amplified genes coding
for 16S rRNA. Appl Environ Microbiol. 1993;59(3):695–700.
Cocolin L, Bisson LF, Mills DA. Direct profiling of the yeast dynamics in wine fermentations. FEMS Microbiol Lett. 2000;189(1):81–7.
Muyzer G. DGGE/TGGE a method for identifying genes from natural ecosystems. Curr Opin Microbiol. 1999;2(3):317–22.
Weerasekera MM, Sissons CH, Wong L, Anderson S, Holmes AR,
Cannon RD. Use of denaturing gradient gel electrophoresis for
the identification of mixed oral yeasts in human saliva. J Med Microbiol. 2013;62(Pt 2):319–30.
Michaelsen A, Pinzari F, Ripka K, Lubitz W, Pinar G. Application of
Jundishapur J Microbiol. 2015;8(10):e22249
Mohammadi P et al.
18.
19.
molecular techniques for identification of fungal communities colonising paper material. Int Biodeter Biodegr. 2006;58(34):133–41.
Kowalchuk G, Smit E. Fungal community analysis using PCRdenaturing gradient gel electrophoresis (DGGE). In: Kowalchuk
GA, Bruijn FJ, Head IM, Akkermans ADL, vanElsas JD, editors. Molecular microbial ecology manual. Dordrech: Kluwer Academic
Publishers; 2004. p. 771–88.
Borresen-Dale AL, Lystad S, Langeroed A. Temporal temperature gradient electrophoresis on the DCode system. Biorad Bull.
Jundishapur J Microbiol. 2015;8(10):e22249
20.
21.
1997;8:2133.
Farnleitner AH, Kreuzinger N, Kavka GG, Grillenberger S, Rath J,
Mach RL. Comparative analysis of denaturing gradient gel electrophoresis and temporal temperature gradient gel electrophoresis in separating Escherichia coli uidA amplicons differing in
single base substitutions. Lett Appl Microbiol. 2000;30(6):427–31.
Marie D, Zhu F, Balague V, Ras J, Vaulot D. Eukaryotic picoplankton communities of the Mediterranean Sea in summer assessed
by molecular approaches (DGGE, TTGE, QPCR). FEMS Microbiol
Ecol. 2006;55(3):403–15.
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